The effect of topography and mechanical stimulation on tenogenic differentiation of human induced pluripotent stem cells

Introduction: The injured tendon can rarely recover to its original structure and function, instead, scar tissue often develops in the defect site. Hence, various strategies for tendon regeneration, involving cells and/or biomaterials, have been explored, to aid the healing process when the natural process fails. Among these approaches culture platforms incorporating topographical cues or inducing mechanical stimulation seem to be promising tools for enhancing the phenotypical, structural and functional aspects of tenocytes. Objectives: The aim of this study is to gain a deeper understanding of how the combination of microgrooved topographies and mechanical stimulation affects the shape and orientation of tendon-derived induced pluripotent stem cells (iPSCs), to engineer a microenvironment where tendon regeneration can be studied. Methods: Microgrooved surfaces were produced in poly(dimethylsiloxane) (PDMS) by soft lithography. Several sizes of grooves and ridges with two different heights were fabricated and characterized by scanning electron microscopy and confocal laser scanning profilometer. After optimizing the surface treatment and coating composition, iPSCs-derived tenocytes were cultured on the treated substrates for up to 7 days on the different microgrooved surfaces. Cell morphology and the number of cell nuclei were evaluated by immunocytochemical staining. Furthermore, the effect of mechanical stimulation will be analyzed on selected substrates. Results and Discussion: Microgrooves with a height of 15-µm were successfully fabricated on PDMS substrates, as opposing to microgrooves with 4-µm height. Fluorescent microscopy images show a more pronounced alignment in iPSC-derived tenocytes along the microgrooves with a height of 15-µm, featuring 3.052-µm-wide grooves and 1.007- µm-ridges. This alignment was indicated by an increase in the form factor descriptor. Furthermore, cells on these patterns expressed scleraxis, a transcription factor characteristic of tenocytes. In conclusion, the microgrooves with a height of 15-µm on PDMS substrates demonstrated a significant impact on the alignment of iPSC-derived tenocytes and sustained the expression of tenogenic markers.